Single-cell transcriptome atlas reveals developmental trajectories and a novel metabolic pathway of catechin esters in tea leaves

Abstract

The tea plant is an economically important woody beverage crop. The unique taste of tea is evoked by certain metabolites, especially catechin esters, whereas their precise formation mechanism in different cell types remains unclear. Here, a fast protoplast isolation method was established and the transcriptional profiles of 16 977 single cells from 1st and 3rd leaves were investigated. We first identified 79 marker genes based on six isolated tissues and constructed a transcriptome atlas, mapped developmental trajectories and further delineated the distribution of different cell types during leaf differentiation and genes associated with cell fate transformation. Interestingly, eight differently expressed genes were found to co-exist at four branch points. Genes involved in the biosynthesis of certain metabolites showed cell- and development-specific characteristics. An unexpected catechin ester glycosyltransferase was characterized for the first time in plants by a gene co-expression network in mesophyll cells. Thus, the first single-cell transcriptional landscape in woody crop leave was reported and a novel metabolism pathway of catechin esters in plants was discovered.

Keywords: ScRNA-seq; catechin; development; glycosyltransferase; leaf; tea plant.

Figure 1

Cell characters of the 1st and 3rd leaves of Camellia sinensis var Shuchazao. (a) The mid‐vein cross section of the 1st leaf. Bar = 200 μm. (b) The leaf lamina cross section of the 1st leaf. VB, vascular bundle. (c) The mid‐vein cross section of the 3rd leaf. (d) The leaf lamina cross section of the 3rd leaf. (e) The lower epidermis of 3rd leaf. Bar = 500 μm. (f) The upper epidermis of 3rd leaf. (g) Contents of main catechins, caffeine and theanine in the 1st and 3rd leaf. (h) Autofluorescent images of the lamina and mid‐vein section of 1st leaf and 3rd leaf. Abbreviation: EM, emission light wavelength; EX, excitation light wavelength LE, lower epidermis; NGT, non‐glandular trichome; PH，phloem; PM, palisade mesophyll; SM, spongy mesophyll; UE, upper epidermis; VBS, vascular bundle sheath; XY, xylem.

Figure 2

Brief flowchart of tea tender leaves scRNA‐seq. The 1st and 3rd leaves of young shoot of C. sinensis var Shuchazao, representing 1 L and 3 L, respectively, were used for single‐cell transcriptomics. Protoplasts were obtained by enzymatic hydrolysis. mRNA released by rupture of a single‐cell suspension was combined with gel bead and emulsion to form GEMs [10× Genomics Chromium Single‐Cell 3′ kit (V3)]. The mRNA of the cell was independently reverse‐transcribed in each GEM, and tagged cDNA was mixed and amplified for library construction. Libraries were sequenced on an Illumina NovaSeq 6000 sequencing system (paired‐end multiplexing run, 150 bp) by LC‐Bio Technology co. ltd., (Hangzhou, China). Six tissues of 3 L were used for cDNA preparation to identify cluster‐specific genes expression pattern by qRT‐PCR. Red arrow: scRNA‐seq analysis; black arrow: experiment validation.

Figure 3

Overviews of the cell atlas of tender tea leaves. (a) Anatomy and cell types of tea 3 L. PM: palisade mesophyll cells; SM: spongy mesophyll cells. (b) Cell distribution in each cluster of 1 L and 3 L. (c) Visualization of 16 cell clusters using tSNE. Dots, individual cells; n = 16 977 cells; colour, cell clusters. (d) Expression pattern of representative cluster‐specific marker genes. Dot diameter, proportion of cluster cells expressing a given gene. The detail information of selected genes is given in Table S8.

Figure 4

Marker genes identification by qRT‐PCR of 6 isolated tissues from 3 L. (a) Heatmap of verified marker gene. The original expression data was given in the Table S9. (b) Identified cell types of 6 isolated tissues under the light microscope. Upper epidermis (UE), lower epidermis (LE), xylem (XY) and phloem and procambia (PH) were stained with safranin O/fast green, which stained lignified cells red and cytoplasm green. Spongy mesophyll cells (SM) and palisade mesophyll cells (PM) were isolated according to published methods and observed directly under the light microscope. (c) tSNE plots with the expression of selected marker genes for six tissues.

Figure 5

Differentiation trajectory and cell fate decision analysed by pseudotime analysis. (a–d) The cell ordering along the differentiation trajectory successively presented by pseudotime states, samples, branch states and cell types. (e) Heatmap of the top 50 significantly changed genes of 9 cell states. The description of these genes is given in Table S10. (f) Representative genes of four branch points were selected to show their expression trends before and after cell differentiation. (g–j) Heatmap of the top 100 significantly changed genes discovered by the Branched expression analysis modelling (BEAM) function from monocle in four branch points. The detail information of these genes is given in Table S11. (k) Brief chart of cell fate decision in the tea tender leaves.

Figure 6

Cell‐specific distribution of genes related to the biosynthesis of lignin and catechins, and discovery of novel glycosylated catechins. (a) Schematic diagram of the flavonoids and lignin biosynthesis pathway. Blue and red molecular structures represent backbones of lignin and catechins, respectively. (b) Heatmap of cell‐specific genes showing of the lignin and catechin biosynthesis pathway. E: epidermis cell; V: vascular bundle cells; M: mesophyll cells; P: proliferating cell. The original data of the heatmap can be found in Table S12. (c) Co‐expression network of UGTs and catechins biosynthesis genes in mesophyll cells. The purple dots present UGTs and the dot radius represents average gene expression level of scRNA‐Seq of mesophyll cells. The yellow dots present catechins biosynthesis related genes. The symbols and spearman correlation coefficients data are given in Table S13. (d) q‐RT‐PCR of UGT72B23 in 6 isolated tissues of 3 L. CsUBI (CSS0007748, ubiquitin‐conjugating enzyme) and CsACTIN (CSS0008920, actin 7 isoform 1) were used as references.

Figure 7

Characterization of UGT72B23 in vivo and in vitro. (a) Activity screening of recombinant CSS0024764 proteins with different substrates. The activity of ECG was set as 100%. Values are expressed as the mean ± standard deviation of triplicate samples. (b) ECG glucoside identification. m/z 605 EIC of tea leaves, (c, d) Kinetic data of recombinant CSS0024764 for ECG and EGCG, respectively. (e) Mass spectral analysis of the formed product rt = 7.62 min, and ESI tandem mass product ions (m/z) of glycoside derivatives of epicatechin gallate following the glucose position in negative mode. (f) Mass spectral of UGT72B23 overexpression in tobacco. (g, i, k) Relative expression level of UGT72B23 in overexpressed tobacco lines, silenced in tea leaves by AsODN and different tissues of tea plant, respectively. (h, j, l) Relative concentration of ECG glucosides in overexpressed tobacco lines, silenced in tea leaves by AsODN and different tissues of tea plant, respectively. Duncan's multiple‐range test was carried out, and statistical significance was calculated with one‐way ANOVA using SPSS 20.0 (P < 0.05). CK, empty vector control; UGT72B23‐OE, overexpression of UGT72B23.

Figure 8

Model for spatiotemporal‐specific UGT72B23‐mediated ECG glucoside formation at single‐cell resolution.